Sickle cell anemia is one of the most commonly inherited genetic diseases, annually affecting approximately 275,000 births worldwide (Modell et al., (2008) Bull World Health Organ.: 86:480-7). The disease is most prevalent in African countries and countries in which a large proportion of the population is of African descent, such as the Caribbean nation of Haiti. In Western Africa, 1.7% of children are born with the disease, and in the Caribbean, the percentage is 0.4%. Id. In particular, the prevalence of sickle hemoglobin (HbS) in Haiti, representing both homozygotes and heterozygotes, is 15.2%. (Randolph (2010) Clin Lab Sci.; 23:79-83)
Sickle cell syndromes result from a single point mutation causing the substitution of valine for glutamic acid at position six of the β-globin chains, producing HbS (α2β2s) instead of normal hemoglobin, HbA (α2β2). (Pauling et al. (1949) Science.; 110:543-8; Marotta et al. (1977) J Biol Chem; 252:5040-53; Ingram (1959) Biochim Biophys Acta.; 36:402-11) The sickle cell β-globin gene must be inherited in the homozygous state for this mutation to result in sickle cell disease (SCD). The heterozygous state produces sickle cell trait (SCT) and few, if any, complications. Other clinical conditions involving the sickle cell gene can occur in individuals inheriting one sickle cell allele with a β-thalassemia allele or another β-chain hemoglobinopathy (compound heterozygotes) (Steinberg. (2008) The Scientific World Journal.; 8:1295-1324).
The clinical symptoms of SCD stem from a change in the intracellular polymerization of HbS when in the deoxyhemoglobin form (Eaton and Hofrichter (1990) Adv Protein Chem.; 40:63-279). This altered polymerization results in sickle-shaped red blood cells (RBCs), which become trapped in the microvasculature and cause hypoxia, tissue death, and the severe pain associated with sickle cell crises. In addition, extravascular and intravascular hemolysis causes hemolytic anemia. A host of other complications are also present that, together, will lead to early death if left untreated. (Steinberg. (2008) The Scientific World Journal.; 8:1295-1324)
Established treatments for SCD involve infection prophylaxis, pain management, and blood transfusion with iron chelation (Ballas Drugs. (2002); 62:1143-72). A more recent approach to treatment involves the drug hydroxyurea to increase expression of fetal hemoglobin (HbF), which proportionately reduces the percentage of HbS. HbF (α2γ2) interrupts HbS polymerization, preventing formation of sickle cells and reducing the frequency of vaso-occlusive events. (Goldberg et al., (1977) J Biol Chem.; 252:3414-21). Hydroxyurea is relatively inexpensive, administered orally, shown to be effective by clinical trials, and is the only HbF-inducing drug currently approved by the U.S. Food and Drug Administration for treatment of SCD. (Charache et al. (1995) N Engl J Med.; 332:1317-22; Ballas et al. (2006) Health Qual Life Outcomes.; 4:59; Steinberg et al. (2003) JAMA; 289:1645-51) However, the drug also presents challenges. Sickle cell patients exhibit a wide range of baseline HbF levels; and when treated with hydroxyurea, the magnitude of their HbF response also varies widely. (Charache et al. (1995) N Engl J Med.; 332:1317-22.; Steinberg et al. (1997) Blood; 89:1078-88) It is estimated that 10-20% of patients show no increase in HbF levels (Steinberg. (2008) The Scientific World Journal.; 8:1295-1324). Since hydroxyurea is mildly carcinogenic, HbF levels must be monitored during treatment to determine the minimum effective dosage or to stop the drug in non-responders. (Ballas Drugs. (2002); 62:1143-72; Ferster et al., (2003) Br J Haematol.; 368-9.)
In developed countries, HbF levels are monitored by electrophoresis or HPLC, but these methods are impractical in underdeveloped countries. Spectrophotometric methods of HbF measurement based upon the resistance of HbF to alkali denaturation have been described, but these methods are limited by method complexity and the need for transport, handling, and storage of a caustic base (NaOH or KOH) and potassium cyanide (KCN). (Singer et al., (1951) Blood; 6:413-28; Betke K, et al., (1959) Nature; 184:1877-8; Pembrey et al., 1972 J Clin Pathol.; 25:738-40; Serjeant et al., 1975 J Clin Pathol.; 28:761-4) One method, the Kleihauer-Betke test (K-B test), is used to determine the presence or absence of HbF-containing RBCs in a blood smear. It is based on the elution of HbA or HbS in citric acid solutions. However, the K-B test is not able to make quantitative determinations of HbF.
The Inventor discloses herein, a simple inexpensive method of monitoring HbA, HbS, or HbF in a quantitative manner, that may be used to monitor patients with sickle cell anemia undergoing hydroxyurea therapy and may be easily practiced in underdeveloped countries,